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Effect of two treatment protocols for ketosis on the resolution, postpartum health, milk yield, and reproductive outcomes of dairy cows
Accepted Manuscript Effect of two treatment protocols for ketosis on the resolution, postpartum health, milk yield, and reproductive outcomes of dairy cows Jae-Kwan Jeong, In-Soo Choi, Sung-Ho Moon, Soo-Chan Lee, Hyun-Gu Kang, Young-Hun Jung, Soo-Bong Park, Ill-Hwa Kim PII:
S0093-691X(17)30460-0
DOI:
10.1016/j.theriogenology.2017.09.030
Reference:
THE 14277
To appear in:
Theriogenology
Received Date: 6 April 2017 Revised Date:
5 August 2017
Accepted Date: 19 September 2017
Please cite this article as: Jeong J-K, Choi I-S, Moon S-H, Lee S-C, Kang H-G, Jung Y-H, Park S-B, Kim I-H, Effect of two treatment protocols for ketosis on the resolution, postpartum health, milk yield, and reproductive outcomes of dairy cows, Theriogenology (2017), doi: 10.1016/ j.theriogenology.2017.09.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised
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Effect of two treatment protocols for ketosis on the resolution,
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postpartum health, milk yield, and reproductive outcomes of dairy
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cows
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Jae-Kwan Jeonga, In-Soo Choia, Sung-Ho Moona, Soo-Chan Leea, Hyun-Gu
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Kanga, Young-Hun Jungb, Soo-Bong Parkb, Ill-Hwa Kima,*
College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 28644, Korea b
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National Institute of Animal Science, RDA, Cheonan, Chungnam, 31000 Korea
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IH Kim
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College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 28644, Korea
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Corresponding author:
Tel.: +82 43 2612571; Fax: +82 43 2673150; E-mail: [email protected].
Jae-Kwan Jeong: [email protected]
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In-Soo Choi: [email protected]
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Sung-Ho Moon: [email protected]
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Soo-Chan Lee: [email protected]
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Hyun-Gu Kang: [email protected]
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Young-Hun Jung: [email protected]
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Soo-Bong Park: [email protected]
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Abstract We determined the effect of ketosis treatment with propylene glycol (PG) or PG plus L-carnitine and
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methionine (Metabolase®, Fatro, Bologna, Italy) on the resolution, postpartum health, milk yield, and
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reproductive performances of dairy cows. Blood from 475 Holstein cows was collected weekly until 4
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weeks after calving to measure blood β-hydroxybutyrate (BHBA) concentrations. Cows with blood
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BHBA concentration ≥ 1.2 mmol/L were diagnosed with ketosis and were enrolled. One hundred and fifty
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cows diagnosed with ketosis were randomly assigned to three treatment groups (Day 0): (1) PG (300 g,
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PO) for 3 days (PG group, n = 50), (2) PG (300 g, PO) plus L-carnitine (1.25 g) plus methionine (5 g, IV)
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for 3 days (PG+CM group, n = 50), and (3) no treatment (control group, n = 50). On Day 3, blood was
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collected to evaluate whether the ketosis had resolved. Cows in the PG and PG+CM groups with blood
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BHBA ≥ 1.2 mmol/L were retreated for an additional 2 days, and then blood BHBA concentration was
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evaluated on Days 5 and 10. Blood glucose and haptoglobin concentrations, rumen fill score (RFS), and
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body condition score (BCS) were measured on Days 0, 3, 5, and 10. Postpartum complications, milk yield
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during the first 2 months, and reproductive outcomes were evaluated. The probability of resolution from
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ketosis was higher (P < 0.05) in the PG+CM group than in the control group on Days 3, 5, and 10 (odds
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ratio: 2.6–6.3). Blood BHBA in the PG+CM group was lower (P < 0.05) than that of the control group on
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Days 3 and 5, whereas blood glucose in the PG+CM group was higher (P < 0.05) than that of the control
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group on Days 3 and 5. RFS in the PG and PG+CM groups was higher than that of the control group on
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Day 10 (P < 0.01), while BCS loss from Day 0 to 10 in the control group was higher than those of the PG
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and PG+CM groups (P < 0.05). Milk yields on the 30th and 60th days postpartum were higher in the
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PG+CM group than the control and PG groups (P < 0.05). Postpartum complications and intervals
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between calving and first postpartum insemination or pregnancy did not differ among the groups (P >
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0.05). In conclusion, treatment of dairy cows with PG plus L-carnitine and methionine improved the
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chances of resolution of ketosis and increased milk yield, while affecting neither the incidence of
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postpartum complications nor reproductive performance.
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Key words: Ketosis; Propylene glycol; Metabolase®; Postpartum health; Milk yield; Dairy cows.
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1. Introduction
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Cows that adapt poorly to negative energy balance (NEB) during their transition period may develop
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hyperketonemia (clinical or subclinical ketosis) within 4 weeks postpartum [1,2]. During this period, a
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large amount of body fat is being used for energy to support milk production, causing a marked
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mobilization of lipids and a pronounced increase in circulating non-esterified fatty acids and ketone
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bodies in tissues and milk [1]. Some cows (2–15%) reduce their dry matter intake (DMI), lose weight,
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and show a decrease in milk yield [3,4], but other cows are affected by subclinical ketosis, defined by an
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elevated concentration of ketone bodies in the absence of clinical signs [5]. However, even subclinical
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ketosis is associated with increases in the incidence of postpartum disorders (including abomasal
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displacement and metritis) and culling, along with decreased milk yield and reproductive performance,
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resulting in severe economic loss [6–9]. Thus, this is a very common and important metabolic disorder in
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modern high productivity dairy herds [4,10].
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Ketosis can be diagnosed by measuring the concentration of ketone bodies (β-hydroxybutyrate
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[BHBA], acetone, and acetoacetate), intermediate metabolites of fatty acid oxidation present in blood,
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urine, or milk. A cow-side handheld meter for the measurement of blood BHBA concentrations has been
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used to rapidly diagnose subclinical ketosis in dairy herds [5,7]. By using this simple device, higher
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incidences of subclinical ketosis have been reported: 6.9–43% in ten European dairy farms [7], 26.4–
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55.7% in four US farms [11], and 13.7–36.1% in four Korean farms [6].
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The earlier diagnosis and effective treatment of subclinical ketosis is challenging but may help
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mitigate further economic loss [12,13]. Supplementation of dairy cows with glucose can be used as a
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treatment for ketosis, resulting in lower levels of ketogenesis in the liver [1,14]. A large number of studies
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have been undertaken to identify effective treatments and evaluate glucose precursors (oral propylene
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glycol [PG]), glucocorticoid, niacin, insulin, recombinant bovine somatotropin, butaphosphan,
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cyanocobalamin, and combined therapies [11,15–19]. However, a fully satisfactory treatment protocol has
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not been established; thus further investigation is required [11,17,20,21].
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Previous studies have shown that PG is readily available in cows and that most of it is relatively
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quickly absorbed from the rumen and utilized in the liver for gluconeogenesis [22–24]. A few studies
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have reported that treatment with PG improves the chances of ketosis resolution, increases milk yield, and
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reduces the incidence of abomasal displacement and culling during the first postpartum month [9,11].
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Another study recommended a treatment protocol for ketosis of 300 mL PG administered orally once
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daily for 5 d [25]. These studies provide a basis for interventions aimed at reducing the negative effects of
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ketosis using PG [9,11,25]. However, other studies have suggested that additional adjunct therapeutic
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agents are required for the effective treatment of ketosis in dairy cows [20,26,27].
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Dairy cows experience a state of reduced liver function coupled with increased inflammation and
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oxidative stress during the peripartum period [28]. Up to 50% of cows have fatty liver (an accumulation
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of triglycerides in hepatocytes) in the first 4 weeks after calving [29] and fatty infiltration represents a
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harmful stimulus to liver parenchymal cells, causing them to release larger amounts of haptoglobin, an
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acute phase protein [30]. Carnitine has an important role in various metabolic functions, including
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mitochondrial long-chain fatty acid oxidation, and has been shown to dramatically ameliorate or prevent
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liver lipid accumulation in dairy cows [31]. Thus, during the transition period carnitine status may
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influence the degree of liver lipid accumulation, while a prepartum increase in liver carnitine level is
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associated with a decrease in liver triglyceride accumulation during this period [32]. In addition, carnitine
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supplementation has been shown to increase hepatic glucose production by stimulating metabolite flux
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through pyruvate carboxylase [33].
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Methionine is an important nutrient involved in the transport of hepatic lipids because it promotes the
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synthesis of phosphatidylcholine to package very low density lipoproteins (VLDL) [34]. Thus, increasing
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the supply of methionine enhances the capacity of liver to export triacylglycerol in the form of VLDL
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[35] and helps ameliorate the negative effects of fatty acid accumulation in the liver after calving [36].
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However, to our knowledge, the effect of combined parenteral therapy with carnitine and methionine on
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ketotic cows has not been determined. Thus, we hypothesized that administration of an injectable
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carnitine and methionine product (Metabolase®, Fatro, Bologna, Italy), combined with oral PG, might
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improve the resolution of ketosis, postpartum health, production, and reproductive outcomes by providing
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a glucose substrate and reducing ketogenesis in the liver. Therefore, this study determined the effect of
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treatment with PG or PG plus L-carnitine and methionine on the resolution of ketosis, blood BHBA,
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glucose and haptoglobin concentrations, postpartum health status (rumen fill, loss of body condition, and
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postpartum complications), milk yield, and reproductive performance in dairy cows.
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2. Materials and methods
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2.1. Animals
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The study was performed on four Holstein dairy farms, located in Chungcheong Province, Korea.
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Each farm contained 80–200 milking cows. The cows were maintained in loose housing systems and fed
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a total mixed ration. The ration was based on brewers grain, alfalfa hay, cotton seed, beet pulp, corn
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silage, tall fescue, timothy hay, minerals, and vitamins, and designed to contain 17–20% crude protein,
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4.8–5.0% crude fat, and 25–30% neutral detergent fiber. A total of 475 Holstein dairy cows, with a
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history of 2.6 ± 1.4 lactations (mean ± standard deviation; range: 1–7 lactations), were included in the
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study. All calved cows were milked twice daily, and milk yield was measured and recorded every day for
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each cow during the first 2 months. All experiments were performed with the approval of the Institutional
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Animal Care and Use Committee of Chungbuk National University, Chungbuk, Korea.
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2. 2. Study design
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Cows with blood BHBA ≥ 1.2 mmol/L were diagnosed with ketosis and enrolled in the study on the
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first occasion this threshold was exceeded until 4 weeks after calving. One hundred and fifty cows
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diagnosed with ketosis were randomly assigned to three treatment groups (Day 0): (1) PG (300 g, PO)
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(500 mL Energyvita, KMBiotech Co. Ltd., Anseong, Korea) for 3 days (PG group, n = 50), (2) PG (300 g,
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PO) plus L-carnitine (1.25 g) and methionine (5 g, IV) (250 mL Metabolase®, Fatro, Bologna, Italy) for 3
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days (PG+CM group, n = 50), and (3) no treatment (control group, n = 50). On Day 3, if the cows in the
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PG and PG+CM groups had BHBA ≥ 1.2 mmol/L, they were retreated for an additional 2 days. The study
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evaluated the effects of each treatment protocol (non-treated controls, PG, or PG plus L-carnitine plus
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methionine product) for ketosis based on the following outcomes: (1) resolution of ketosis, based on
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blood BHBA < 1.2 mmol/L on Days 3, 5, and 10; (2) blood BHBA, glucose, and haptoglobin
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concentrations and RFS on Days 0, 3, 5, and 10; (3) loss of BCS from Day 0 to 10; (4) occurrence of
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postpartum complications (including abomasal displacement, metritis, endometritis, pyometra, digestive
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disorders, mastitis, and lameness) during the first postpartum month; (5) milk yield, defined as the
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average yield on the 30th and 60th days postpartum; and (6) reproductive performance (interval from
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calving to first postpartum insemination or pregnancy).
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2. 3 Blood sampling, and measurement of blood metabolites and serum haptoglobin
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Blood from 475 Holstein cows was collected from the tail vein weekly until 4 weeks after calving to
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measure BHBA concentrations in whole blood using an electronic handheld meter and β-ketone test strips
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(FreeStyle Optimum, Abbott Diabetes Care Ltd., Witney, UK). On Days 0, 3, 5, and 10, blood BHBA and
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glucose concentrations were measured using the electronic handheld meter and FreeStyle Optimum blood
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β-ketone and glucose test strips (Abbott Diabetes Care Ltd., Witney, UK). In addition, on each of Days 0,
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3, 5, and 10, 10 mL blood was placed into plain plastic centrifuge tubes and immediately placed in an ice
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bath. These samples were then centrifuged at 2000 × g for 10 min at 4°C, and the serum was harvested
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and frozen at -80°C until assayed for serum haptoglobin.
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Haptoglobin concentration was determined using a commercially available bovine haptoglobin ELISA
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test kit (Life Diagnostics, Inc., West Chester, PA, USA). All procedures were performed according to the
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guidelines provided by the manufacturer. The intra- and inter-assay coefficients of variation were 3.1%
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and 6.7%, respectively.
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2. 4. Assessment of postpartum health and reproductive management
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Rumen fill score (RFS) and body condition score (BCS) were evaluated on Days 0, 3, 5, and 10. RFS
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was measured 2 h before feeding on a 5-point scale (1 = insufficient feed intake and 5 = sufficient feed
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intake) using a visual technique [37]. At the same time, BCS was measured on a 5-point scale (with
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quarter-point division) using a visual technique [38].
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The definitions used for postpartum disorders used in the present study were similar to those
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described in previous publications [39–41]. Abomasal displacement was diagnosed by a ‘ping’ sound
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during abdominal auscultation. Metritis was defined by the presence of fever (≥39.5°C) and a watery,
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fetid uterine discharge. Pyometra was defined by the detection of a uterus distended with pus during
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ultrasonography. Endometritis was diagnosed at 4 weeks postpartum by examining any vaginal discharge
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sampled using the Metricheck tool [42]. Briefly, after cleaning the vulva with the disinfectant
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chlorhexidine gluconate, the Metricheck device was inserted until it reached the vaginal fornix and then
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retracted for evaluation of the vaginal mucus contained in the cup. Cows with a mucopurulent uterine
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discharge (>50% pus) were diagnosed with endometritis. Digestive problems were diagnosed by the
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presence of diarrhea or bloat, and mastitis by the presence of abnormal milk or signs of inflammation in
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one or more quarters of the udder. Lameness was diagnosed based on abnormal gait or lack of weight
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bearing on a limb, and included diagnoses of interdigital and digital dermatitis. In this study, the voluntary waiting period from calving to the first artificial insemination (AI) was 45
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days. In addition to estrus detection, a herd reproductive management program was employed. Estrus
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synchronization was achieved with prostaglandin F2α (PGF2α) or Ovsynch [43]. Ovsynch synchronization
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was performed with a combination of GnRH on Day 0, PGF2α on Day 7, and GnRH on Day 9. Cows that
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exhibited estrus naturally or after estrus synchronization using PGF2α were inseminated according to the
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am-pm rule. Cows treated with Ovsynch received timed AI. Pregnancy diagnosis was performed at 40–50
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days post AI by transrectal palpation and ultrasonography. Reproductive performance data were collected
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for a minimum of 210 days postpartum or until pregnancy or culling.
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2.5. Statistical analyses
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Data were expressed as the mean ± SEM. Statistical analyses were performed using the SAS program
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(version 9.4, SAS Inst., Cary, NC, USA). For statistical analyses, cows were categorized as either
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primiparous or multiparous, and the calving season was defined as spring (March to May), summer (June
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to August), autumn (September to November), or winter (December to February). Enrollment week was
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categorized as 1 or ≤2.
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To determine the probability of ketosis resolution on Days 3, 5, and 10, and the incidence of
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postpartum complications during the first postpartum month, logistic regression was undertaken using the
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LOGISTIC procedure. To determine the probability of ketosis resolution, the logistic regression model
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included the farm, cow parity, enrollment week, treatment group (control, PG, and PG+CM), and
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interactions between these variables. To determine the probability of the occurrence of postpartum
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complications, the logistic regression model included the farm, calving season, cow parity, treatment
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group, and interactions between these variables. Backward stepwise regression was used in all models,
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and elimination was performed based on the Wald statistic criterion when P > 0.15. The odds ratio (OR)
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and 95% confidence interval (CI) were calculated. Data are presented as proportions and ORs with their
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respective 95% CIs.
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The blood BHBA, glucose and haptoglobin concentrations, RFS, and milk yield were analyzed using
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mixed models. The statistical models included group effects (control, PG, and PG+CM), cow parity
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(primiparous or multiparous), measuring time (Days 0, 3, 5, and 10), and two-way interactions between
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group, cow parity, and measuring time. Haptoglobin concentrations were not normally distributed;
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therefore, values were transformed to their natural logarithms for data analysis, although non-transformed
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data expressed as means and SEM are presented herein. For milk yield, the statistical models included
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group effects (control, PG, and PG+CM), measuring time (Days 30 and 60 postpartum), and two-way
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interactions between groups, cow parity, and measuring time. Cows were included in the model as a
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random effect. ANOVA followed by Duncan’s multiple range test was performed when either a group
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effect or an interaction between group and measuring time was observed. Loss of BCS from Day 0 to 10
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was analyzed by ANOVA followed by Duncan's multiple range test.
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Cox’s proportional hazard model with the PHREG procedure was used to analyze the chance of first
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insemination by 150 days and pregnancy by 210 days postpartum among the control, PG, and PG+CM
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groups. This estimated the chance of a cow having been inseminated or being pregnant at a given time.
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The time variables used in this model were the interval in days between calving and first insemination,
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and between calving and pregnancy. Cows that died, were sold, had not been inseminated by 150 days
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postpartum, or were not pregnant by 210 days postpartum were not included in the analysis. Cox models
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included farm, cow parity (primiparous or multiparous), enrollment week (1 or ≤2), and group (control,
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PG, and PG+CM). The proportional hazard rate was determined based on interactions between
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explanatory variables and time, and by evaluating Kaplan–Meier curves. The median and mean days to
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first insemination, or days to pregnancy, were determined by survival analysis using the Kaplan–Meier
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model and the LIFETEST procedure within SAS software. P < 0.05 was considered significant.
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3. Results
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The effect of the treatment protocols on the probability of resolution of ketosis on Days 3, 5, and 10
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was determined using the logistic regression model (Table 1). The probability of ketosis resolution was
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higher in the PG+CM group on Days 3 (OR: 6.3, P < 0.001), 5 (OR: 2.6, P < 0.05), and 10 (OR: 3.6, P <
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0.01), respectively, than in the control group, whereas the probability of resolution in the PG group was
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intermediate (P > 0.05). However, the logistic regression model revealed that the probability of resolution
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was not associated with farm, cow parity or enrollment week (P > 0.05).
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There was no effect of group on blood BHBA concentration (P > 0.05), but there were significant
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effects of measuring time (P < 0.0001) and an interaction between group and measuring time (P < 0.05)
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(Fig. 1). The BHBA concentration in the PG+CM group was lower than that of the control group at Days
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3 (P < 0.01) and 5 (P < 0.05), whereas the BHBA concentration in the PG group was intermediate (P >
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0.05). There was no parity effect on blood BHBA concentration. There was no effect of group on blood
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glucose (P > 0.05), but there were significant effects of measuring time (P < 0.01) and an interaction
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between group and measuring time (P = 0.05) (Fig. 2). The glucose concentrations in the PG and PG+CM
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group were higher than that of the control group at Day 3 (P < 0.01), while the glucose concentration in
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the PG+CM group was higher than that of the control group at Day 5 (P < 0.05). There was no parity
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effect on blood glucose concentration. There was a significant effect of measuring time on serum
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haptoglobin concentration (P < 0.0001), but no effect of group (ranges: 98.5 ± 32.3–120.0 ± 29.1 µg/mL)
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or any interaction between group and measuring time (P > 0.05) (data not shown). There was no effect of
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parity on serum haptoglobin concentration.
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There was a significant effect of group on RFS (P < 0.05), but there was no effect of measuring time
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or the interaction between group and measuring time (P > 0.05) (Fig. 3). The RFS in the PG group was
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higher than that of the control group on Day 5 (P < 0.05), whereas the RFSs in the PG and PG+CM
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groups were higher than that of the control group at Day 10 (P < 0.01). Multiparous cows had lower RFS
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than primiparous cows (P < 0.05). BCS loss from Day 0 to 10 in the control group (0.19 ± 0.02) was
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higher (P < 0.05) than those of the PG and PG+CM groups (each 0.12 ± 0.02).
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There were significant effects of group on milk yield (P < 0.05) and measuring time (P < 0.001), but
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no interaction between group and measuring time (P > 0.05) (Fig. 4). The average yield in the PG+CM
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group was greater than those of the control and PG groups on the 30th and 60th days postpartum (P <
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0.05). Primiparous cows showed lower milk yield than multiparous cows (P < 0.001). We determined the
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effect of treatment protocols for ketosis on the probability of occurrence of postpartum complications
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using the logistic regression model. Neither the treatment protocol (range: 48.0–54.0%), farm, calving
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season, and parity affected the incidence of postpartum complications (P > 0.05).
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The intervals between calving and first postpartum insemination or pregnancy did not differ among
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the groups (P > 0.05). The median and mean days to first postpartum insemination were 80 and 92.6 ± 4.7
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in the control group, 84 and 99.1 ± 5.5 in the PG group, and 97 and 103.1 ± 4.7 in the PG+CM group,
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respectively. The median and mean days to pregnancy were 154 and 152.4 ± 7.3 in the control group, 160
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and 154.7 ± 8.7 in the PG group, and 169 and 156.3 ± 8.1 in the PG+CM group, respectively.
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4. Discussion
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We have evaluated the effect of treatment with PG or PG plus an L-carnitine and methionine-
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containing product (Metabolase®, Fatro, Bologna, Italy) on the resolution of ketosis, blood BHBA,
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glucose, and haptoglobin concentrations, postpartum health (RFS, BCS loss, and complications), milk
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yield, and reproductive performance in dairy cows. Treatment with PG plus L-carnitine and methionine
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resulted in faster resolution of ketosis, a lower BHBA and higher glucose concentrations, a higher RFS,
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and lower BCS loss, resulting in higher milk yield, while it did not affect serum haptoglobin
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concentration, incidence of postpartum complications, or reproductive performance.
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Cows treated with PG plus L-carnitine and methionine had a higher probability of ketosis resolution
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on Days 3, 5, and 10, whereas those that received only PG were not more likely to recover from ketosis
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than controls on this timescale. The higher probability of ketosis resolution in PG+CM cows was
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associated with improvements in other indicators of health, including lower blood BHBA and higher
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glucose concentrations, higher RFS, and less BCS loss during the study period. It is likely that the
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supplementation of L-carnitine and methionine, in addition to PG, not only increased hepatic glucose
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production by stimulating metabolite flux through pyruvate carboxylase, but also enhanced the capacity
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of the liver to export triacylglycerol in the form of VLDL [31,33,35].
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Fatty infiltration represents a harmful stimulus to liver parenchymal cells, and consequently
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production of haptoglobin, acute phase protein, may be higher in affected hepatocytes [30]. Consistent
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with this, a previous study showed that haptoglobin increased in cows with ketosis (by 4–6 fold)
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compared with their healthy counterparts during the transition period [44]. However, we found no effect
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of either PG or PG plus L-carnitine and methionine on serum haptoglobin concentration. This may be
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because postpartum disorders are often associated with acute inflammatory processes, including dystocia,
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retained placenta, or metritis, which would confound the interpretation of haptoglobin concentration
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among the groups in the present study. Indeed, several previous studies have demonstrated that such
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postpartum disorders were associated with elevated haptoglobin concentrations [45–47]. However, other
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studies have shown that oral PG increases blood glucose and insulin, but decreases BHBA and NEFA
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concentrations [24,48]. Moreover, improved resolution of ketosis and lower incidence of postpartum
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disease (abomasal displacement) and culling [9,11] have also been reported, which are not consistent with
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our data. The reason for the discrepancies among these studies is not clear; therefore, further work is
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required to resolve these. Monitoring of the changes in feed intake during the periparturient period is very important in dairy
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cows because suboptimal DMI is associated with several postpartum disorders, including ketosis, metritis,
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and mastitis [49–51]. However, the monitoring of DMI for individual cows requires enormous labor and
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is impractical for commercial dairy farms. Thus, the measurement of rumen fill is used as an indirect tool
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for the evaluation of feed intake status. This is a relatively simple method that is feasible for commercial
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dairy farms to undertake [37,52]. In addition, BCS is a simple, indirect assessment tool for the monitoring
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of energy balance. BCS at calving and the change in BCS during the dry period were associated with the
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incidence of postpartum diseases and subsequent reproductive performance in dairy cows [53–55].
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Therefore, we used RFS and the loss of BCS as indicators for feed intake status and energy balance in the
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present study.
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Higher RFS in the PG and PG+CM groups than in the control group at Day 10 imply that cows that
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received two treatments had eaten more than the non-treated control cows. In addition, lower loss of BCS
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during the experimental period (Days 0–10) in the two treatment groups suggests that the extent of their
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NEB had been alleviated by treatment with PG or PG plus L-carnitine and methionine. Taken together,
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our data indicate that a higher RFS and lower BCS loss, as well as decreased blood BHBA and increased
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glucose concentrations, which may imply higher feed intake and lower NEB, might contribute to a higher
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incidence of ketosis resolution in cows that receive PG plus L-carnitine and methionine.
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Previous publications have shown decreases in milk production in cows with ketosis [8,9,56]. Thus,
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early and effective treatment for ketosis may prevent a subsequent decrease in milk production. Cows
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treated with PG plus L-carnitine and methionine had higher milk yields at Days 30 and 60 postpartum
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than those treated with only PG, or controls, in this study. The positive effect on milk yield in the PG+CM
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group might be a consequence of the earlier resolution of ketosis and better health status compared to the
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other two groups, reflected in higher RFS and lower loss of BCS. Moreover, our results are consistent
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with a previous study in which milk yield was similar between cows that received PG orally and untreated
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control cows [22]. However, another previous study reported increased milk yield in cows treated with
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300 mL PG orally in the first month of lactation [11], which is inconsistent with our data. We found no effects of PG or PG plus L-carnitine and methionine on the incidence of postpartum
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complications and reproductive outcomes (intervals from calving to first postpartum insemination and
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pregnancy). Our results are broadly similar to those of a previous study, in which oral PG administration
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did not affect the incidence of clinical ketosis compared to untreated control cows [22]. Inconsistent with
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our data, oral PG administration decreased the risk of developing an abomasal displacement in a previous
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study [9]. However, this study found no difference in the calving to conception interval between cows that
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received PG and untreated cows [9], which is similar to our results.
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Taken together, our data show that cows treated with PG plus L-carnitine and methionine for ketosis
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resulted in earlier resolution and higher milk yield compared to untreated controls and cows treated with
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PG only. This could be attributed to the provision of a better glucose substrate and the reduction of
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ketogenesis in the liver, as suggested in the Introduction. Moreover, the beneficial outcomes described in
338
the present study might also be due to the increase in postpartum health resulting from increased feed
339
intake and lower NEB, as demonstrated not only by the higher RFS and lower loss of BCS, but also by
340
the decreased blood BHBA and increased glucose concentrations. However, we found no effects of
341
treatment with PG plus L-carnitine and methionine on the incidence of postpartum complications and
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reproductive outcomes. The results showing earlier resolution from ketosis and higher milk yield in cows
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treated with PG plus L-carnitine and methionine might be helpful in guiding treatments for ketotic dairy
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cows, which in turn could potentially mitigate the economic loss associated with ketosis.
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Acknowledgments
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This work was carried out with the support of the “Cooperative Research Program for Agriculture
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Science & Technology Development (Project No. PJ01081802)” Rural Development Administration,
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Republic of Korea.
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Conflicts of interest
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None.
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Figure captions
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Figure 1. Mean (± SEM) blood β-hydroxybutyrate (BHBA) concentrations in the control, PG (propylene
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glycol), and PG+CM (L-carnitine and methionine) group cows (n = 50 per group) at Days 0, 3, 5, and 10
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after treatment. G: group effect, Day: measuring time effect. G*Day: group-by-measuring time effect.
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a,b
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superscripts differ with P < 0.01 among groups.
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c,d
Means with different
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Means with different superscripts differ with P < 0.05 among groups.
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Figure 2. Mean (± SEM) blood glucose concentrations in the control, PG, and PG+CM group cows (n =
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50 per group) at Days 0, 3, 5, and 10 after treatment. G: group effect, Day: measuring time effect. G*Day:
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group-by-measuring time effect.
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c,d
a,b
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Means with different superscripts differ with P < 0.05 among groups.
Means with different superscripts differ with P < 0.01 among groups.
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Figure 3. Mean (± SEM) rumen fill scores in the control, PG, and PG+CM group cows (n = 50 per group)
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at Days 0, 3, 5, and 10 after treatment. G: group effect, Day: measuring time effect. G*Day: group-by-
516
measuring time effect.
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with different superscripts differ with P < 0.01 among groups.
c,d
Means
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Means with different superscripts differ with P < 0.05 among groups.
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a,b
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Figure 4. Mean (± SEM) milk yield in the control (n = 49), PG (n = 48), and PG+CM (n = 48) groups at
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the 30th and 60th days postpartum. G: group effect, Day: measuring time effect. G*Day: group-by-
521
measuring time effect. a,bMeans with different superscripts differ with P < 0.05 among groups.
522 523 524 525
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Table 1. The effect of treatment protocols for ketosis on the probability of resolution of ketosis on Days 3,
527
5, and 10 determined using a logistic regression model. Variable
Resolution a
evaluation
percentage (n )
b
Control
12.0% (6/50)
Reference
PG
22.0% (11/50)
2.1
PG+CM
46.0% (23/50)
6.3
Farm d
Cow parity
e
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531 532
> 0.05
0.509–2.925
> 0.05
PG+CM
48.0% (24/50)
2.6
1.133–6.090
< 0.05
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Group
> 0.05 > 0.05 > 0.05
Reference
44.0% (22/50)
2.2
0.920–5.148
> 0.05
56.0% (28/50)
3.6
1.496–8.464
< 0.01
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30.0% (15/50)
> 0.05
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Farm
c
> 0.05
1.2
PG+CM
530
> 0.05
30.0% (15/50)
PG
Cow parity
> 0.05
Enrollment week
> 0.05
, cases/group total.
b
< 0.001
PG
Control
529
2.256–17.291
Reference
Enrollment week
a
> 0.05
26.0% (13/50)
Cow parity
528
0.699–6.115
Control
Farm
Day 10
P-value
OR
Group
Day 5
95% CI
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Day 3
c
Adjusted
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Days of
, odds ratio.
, confidence interval.
d
, categorized as either primiparous or multiparous.
e
, divided into 1 or ≤2.
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Highlights
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- We determined the effect of ketosis treatment with propylene glycol (PG) or PG plus L-carnitine and
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methionine.
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- PG plus L-carnitine and methionine improved the chances of resolution of ketosis and increased milk
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yield.
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- Outcomes might be associated with better postpartum health due to increased feed intake and lower
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NEB.
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S0093-691X(17)30460-0
DOI:
10.1016/j.theriogenology.2017.09.030
Reference:
THE 14277
To appear in:
Theriogenology
Received Date: 6 April 2017 Revised Date:
5 August 2017
Accepted Date: 19 September 2017
Please cite this article as: Jeong J-K, Choi I-S, Moon S-H, Lee S-C, Kang H-G, Jung Y-H, Park S-B, Kim I-H, Effect of two treatment protocols for ketosis on the resolution, postpartum health, milk yield, and reproductive outcomes of dairy cows, Theriogenology (2017), doi: 10.1016/ j.theriogenology.2017.09.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised
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Effect of two treatment protocols for ketosis on the resolution,
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postpartum health, milk yield, and reproductive outcomes of dairy
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cows
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Jae-Kwan Jeonga, In-Soo Choia, Sung-Ho Moona, Soo-Chan Leea, Hyun-Gu
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Kanga, Young-Hun Jungb, Soo-Bong Parkb, Ill-Hwa Kima,*
College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 28644, Korea b
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National Institute of Animal Science, RDA, Cheonan, Chungnam, 31000 Korea
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*
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IH Kim
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College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 28644, Korea
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Corresponding author:
Tel.: +82 43 2612571; Fax: +82 43 2673150; E-mail: [email protected].
Jae-Kwan Jeong: [email protected]
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In-Soo Choi: [email protected]
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Sung-Ho Moon: [email protected]
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Soo-Chan Lee: [email protected]
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Hyun-Gu Kang: [email protected]
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Young-Hun Jung: [email protected]
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Soo-Bong Park: [email protected]
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Abstract We determined the effect of ketosis treatment with propylene glycol (PG) or PG plus L-carnitine and
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methionine (Metabolase®, Fatro, Bologna, Italy) on the resolution, postpartum health, milk yield, and
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reproductive performances of dairy cows. Blood from 475 Holstein cows was collected weekly until 4
30
weeks after calving to measure blood β-hydroxybutyrate (BHBA) concentrations. Cows with blood
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BHBA concentration ≥ 1.2 mmol/L were diagnosed with ketosis and were enrolled. One hundred and fifty
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cows diagnosed with ketosis were randomly assigned to three treatment groups (Day 0): (1) PG (300 g,
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PO) for 3 days (PG group, n = 50), (2) PG (300 g, PO) plus L-carnitine (1.25 g) plus methionine (5 g, IV)
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for 3 days (PG+CM group, n = 50), and (3) no treatment (control group, n = 50). On Day 3, blood was
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collected to evaluate whether the ketosis had resolved. Cows in the PG and PG+CM groups with blood
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BHBA ≥ 1.2 mmol/L were retreated for an additional 2 days, and then blood BHBA concentration was
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evaluated on Days 5 and 10. Blood glucose and haptoglobin concentrations, rumen fill score (RFS), and
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body condition score (BCS) were measured on Days 0, 3, 5, and 10. Postpartum complications, milk yield
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during the first 2 months, and reproductive outcomes were evaluated. The probability of resolution from
40
ketosis was higher (P < 0.05) in the PG+CM group than in the control group on Days 3, 5, and 10 (odds
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ratio: 2.6–6.3). Blood BHBA in the PG+CM group was lower (P < 0.05) than that of the control group on
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Days 3 and 5, whereas blood glucose in the PG+CM group was higher (P < 0.05) than that of the control
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group on Days 3 and 5. RFS in the PG and PG+CM groups was higher than that of the control group on
44
Day 10 (P < 0.01), while BCS loss from Day 0 to 10 in the control group was higher than those of the PG
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and PG+CM groups (P < 0.05). Milk yields on the 30th and 60th days postpartum were higher in the
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PG+CM group than the control and PG groups (P < 0.05). Postpartum complications and intervals
47
between calving and first postpartum insemination or pregnancy did not differ among the groups (P >
48
0.05). In conclusion, treatment of dairy cows with PG plus L-carnitine and methionine improved the
49
chances of resolution of ketosis and increased milk yield, while affecting neither the incidence of
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postpartum complications nor reproductive performance.
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Key words: Ketosis; Propylene glycol; Metabolase®; Postpartum health; Milk yield; Dairy cows.
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1. Introduction
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Cows that adapt poorly to negative energy balance (NEB) during their transition period may develop
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hyperketonemia (clinical or subclinical ketosis) within 4 weeks postpartum [1,2]. During this period, a
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large amount of body fat is being used for energy to support milk production, causing a marked
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mobilization of lipids and a pronounced increase in circulating non-esterified fatty acids and ketone
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bodies in tissues and milk [1]. Some cows (2–15%) reduce their dry matter intake (DMI), lose weight,
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and show a decrease in milk yield [3,4], but other cows are affected by subclinical ketosis, defined by an
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elevated concentration of ketone bodies in the absence of clinical signs [5]. However, even subclinical
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ketosis is associated with increases in the incidence of postpartum disorders (including abomasal
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displacement and metritis) and culling, along with decreased milk yield and reproductive performance,
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resulting in severe economic loss [6–9]. Thus, this is a very common and important metabolic disorder in
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modern high productivity dairy herds [4,10].
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Ketosis can be diagnosed by measuring the concentration of ketone bodies (β-hydroxybutyrate
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[BHBA], acetone, and acetoacetate), intermediate metabolites of fatty acid oxidation present in blood,
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urine, or milk. A cow-side handheld meter for the measurement of blood BHBA concentrations has been
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used to rapidly diagnose subclinical ketosis in dairy herds [5,7]. By using this simple device, higher
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incidences of subclinical ketosis have been reported: 6.9–43% in ten European dairy farms [7], 26.4–
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55.7% in four US farms [11], and 13.7–36.1% in four Korean farms [6].
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The earlier diagnosis and effective treatment of subclinical ketosis is challenging but may help
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mitigate further economic loss [12,13]. Supplementation of dairy cows with glucose can be used as a
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treatment for ketosis, resulting in lower levels of ketogenesis in the liver [1,14]. A large number of studies
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have been undertaken to identify effective treatments and evaluate glucose precursors (oral propylene
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glycol [PG]), glucocorticoid, niacin, insulin, recombinant bovine somatotropin, butaphosphan,
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cyanocobalamin, and combined therapies [11,15–19]. However, a fully satisfactory treatment protocol has
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not been established; thus further investigation is required [11,17,20,21].
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Previous studies have shown that PG is readily available in cows and that most of it is relatively
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quickly absorbed from the rumen and utilized in the liver for gluconeogenesis [22–24]. A few studies
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have reported that treatment with PG improves the chances of ketosis resolution, increases milk yield, and
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reduces the incidence of abomasal displacement and culling during the first postpartum month [9,11].
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Another study recommended a treatment protocol for ketosis of 300 mL PG administered orally once
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daily for 5 d [25]. These studies provide a basis for interventions aimed at reducing the negative effects of
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ketosis using PG [9,11,25]. However, other studies have suggested that additional adjunct therapeutic
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agents are required for the effective treatment of ketosis in dairy cows [20,26,27].
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Dairy cows experience a state of reduced liver function coupled with increased inflammation and
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oxidative stress during the peripartum period [28]. Up to 50% of cows have fatty liver (an accumulation
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of triglycerides in hepatocytes) in the first 4 weeks after calving [29] and fatty infiltration represents a
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harmful stimulus to liver parenchymal cells, causing them to release larger amounts of haptoglobin, an
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acute phase protein [30]. Carnitine has an important role in various metabolic functions, including
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mitochondrial long-chain fatty acid oxidation, and has been shown to dramatically ameliorate or prevent
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liver lipid accumulation in dairy cows [31]. Thus, during the transition period carnitine status may
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influence the degree of liver lipid accumulation, while a prepartum increase in liver carnitine level is
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associated with a decrease in liver triglyceride accumulation during this period [32]. In addition, carnitine
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supplementation has been shown to increase hepatic glucose production by stimulating metabolite flux
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through pyruvate carboxylase [33].
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Methionine is an important nutrient involved in the transport of hepatic lipids because it promotes the
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synthesis of phosphatidylcholine to package very low density lipoproteins (VLDL) [34]. Thus, increasing
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the supply of methionine enhances the capacity of liver to export triacylglycerol in the form of VLDL
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[35] and helps ameliorate the negative effects of fatty acid accumulation in the liver after calving [36].
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However, to our knowledge, the effect of combined parenteral therapy with carnitine and methionine on
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ketotic cows has not been determined. Thus, we hypothesized that administration of an injectable
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carnitine and methionine product (Metabolase®, Fatro, Bologna, Italy), combined with oral PG, might
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improve the resolution of ketosis, postpartum health, production, and reproductive outcomes by providing
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a glucose substrate and reducing ketogenesis in the liver. Therefore, this study determined the effect of
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treatment with PG or PG plus L-carnitine and methionine on the resolution of ketosis, blood BHBA,
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glucose and haptoglobin concentrations, postpartum health status (rumen fill, loss of body condition, and
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postpartum complications), milk yield, and reproductive performance in dairy cows.
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2. Materials and methods
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2.1. Animals
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The study was performed on four Holstein dairy farms, located in Chungcheong Province, Korea.
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Each farm contained 80–200 milking cows. The cows were maintained in loose housing systems and fed
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a total mixed ration. The ration was based on brewers grain, alfalfa hay, cotton seed, beet pulp, corn
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silage, tall fescue, timothy hay, minerals, and vitamins, and designed to contain 17–20% crude protein,
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4.8–5.0% crude fat, and 25–30% neutral detergent fiber. A total of 475 Holstein dairy cows, with a
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history of 2.6 ± 1.4 lactations (mean ± standard deviation; range: 1–7 lactations), were included in the
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study. All calved cows were milked twice daily, and milk yield was measured and recorded every day for
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each cow during the first 2 months. All experiments were performed with the approval of the Institutional
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Animal Care and Use Committee of Chungbuk National University, Chungbuk, Korea.
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2. 2. Study design
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Cows with blood BHBA ≥ 1.2 mmol/L were diagnosed with ketosis and enrolled in the study on the
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first occasion this threshold was exceeded until 4 weeks after calving. One hundred and fifty cows
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diagnosed with ketosis were randomly assigned to three treatment groups (Day 0): (1) PG (300 g, PO)
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(500 mL Energyvita, KMBiotech Co. Ltd., Anseong, Korea) for 3 days (PG group, n = 50), (2) PG (300 g,
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PO) plus L-carnitine (1.25 g) and methionine (5 g, IV) (250 mL Metabolase®, Fatro, Bologna, Italy) for 3
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days (PG+CM group, n = 50), and (3) no treatment (control group, n = 50). On Day 3, if the cows in the
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PG and PG+CM groups had BHBA ≥ 1.2 mmol/L, they were retreated for an additional 2 days. The study
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evaluated the effects of each treatment protocol (non-treated controls, PG, or PG plus L-carnitine plus
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methionine product) for ketosis based on the following outcomes: (1) resolution of ketosis, based on
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blood BHBA < 1.2 mmol/L on Days 3, 5, and 10; (2) blood BHBA, glucose, and haptoglobin
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concentrations and RFS on Days 0, 3, 5, and 10; (3) loss of BCS from Day 0 to 10; (4) occurrence of
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postpartum complications (including abomasal displacement, metritis, endometritis, pyometra, digestive
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disorders, mastitis, and lameness) during the first postpartum month; (5) milk yield, defined as the
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average yield on the 30th and 60th days postpartum; and (6) reproductive performance (interval from
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calving to first postpartum insemination or pregnancy).
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2. 3 Blood sampling, and measurement of blood metabolites and serum haptoglobin
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Blood from 475 Holstein cows was collected from the tail vein weekly until 4 weeks after calving to
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measure BHBA concentrations in whole blood using an electronic handheld meter and β-ketone test strips
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(FreeStyle Optimum, Abbott Diabetes Care Ltd., Witney, UK). On Days 0, 3, 5, and 10, blood BHBA and
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glucose concentrations were measured using the electronic handheld meter and FreeStyle Optimum blood
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β-ketone and glucose test strips (Abbott Diabetes Care Ltd., Witney, UK). In addition, on each of Days 0,
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3, 5, and 10, 10 mL blood was placed into plain plastic centrifuge tubes and immediately placed in an ice
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bath. These samples were then centrifuged at 2000 × g for 10 min at 4°C, and the serum was harvested
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and frozen at -80°C until assayed for serum haptoglobin.
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Haptoglobin concentration was determined using a commercially available bovine haptoglobin ELISA
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test kit (Life Diagnostics, Inc., West Chester, PA, USA). All procedures were performed according to the
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guidelines provided by the manufacturer. The intra- and inter-assay coefficients of variation were 3.1%
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and 6.7%, respectively.
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2. 4. Assessment of postpartum health and reproductive management
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Rumen fill score (RFS) and body condition score (BCS) were evaluated on Days 0, 3, 5, and 10. RFS
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was measured 2 h before feeding on a 5-point scale (1 = insufficient feed intake and 5 = sufficient feed
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intake) using a visual technique [37]. At the same time, BCS was measured on a 5-point scale (with
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quarter-point division) using a visual technique [38].
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The definitions used for postpartum disorders used in the present study were similar to those
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described in previous publications [39–41]. Abomasal displacement was diagnosed by a ‘ping’ sound
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during abdominal auscultation. Metritis was defined by the presence of fever (≥39.5°C) and a watery,
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fetid uterine discharge. Pyometra was defined by the detection of a uterus distended with pus during
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ultrasonography. Endometritis was diagnosed at 4 weeks postpartum by examining any vaginal discharge
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sampled using the Metricheck tool [42]. Briefly, after cleaning the vulva with the disinfectant
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chlorhexidine gluconate, the Metricheck device was inserted until it reached the vaginal fornix and then
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retracted for evaluation of the vaginal mucus contained in the cup. Cows with a mucopurulent uterine
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discharge (>50% pus) were diagnosed with endometritis. Digestive problems were diagnosed by the
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presence of diarrhea or bloat, and mastitis by the presence of abnormal milk or signs of inflammation in
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one or more quarters of the udder. Lameness was diagnosed based on abnormal gait or lack of weight
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bearing on a limb, and included diagnoses of interdigital and digital dermatitis. In this study, the voluntary waiting period from calving to the first artificial insemination (AI) was 45
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days. In addition to estrus detection, a herd reproductive management program was employed. Estrus
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synchronization was achieved with prostaglandin F2α (PGF2α) or Ovsynch [43]. Ovsynch synchronization
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was performed with a combination of GnRH on Day 0, PGF2α on Day 7, and GnRH on Day 9. Cows that
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exhibited estrus naturally or after estrus synchronization using PGF2α were inseminated according to the
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am-pm rule. Cows treated with Ovsynch received timed AI. Pregnancy diagnosis was performed at 40–50
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days post AI by transrectal palpation and ultrasonography. Reproductive performance data were collected
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for a minimum of 210 days postpartum or until pregnancy or culling.
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2.5. Statistical analyses
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Data were expressed as the mean ± SEM. Statistical analyses were performed using the SAS program
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(version 9.4, SAS Inst., Cary, NC, USA). For statistical analyses, cows were categorized as either
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primiparous or multiparous, and the calving season was defined as spring (March to May), summer (June
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to August), autumn (September to November), or winter (December to February). Enrollment week was
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categorized as 1 or ≤2.
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To determine the probability of ketosis resolution on Days 3, 5, and 10, and the incidence of
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postpartum complications during the first postpartum month, logistic regression was undertaken using the
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LOGISTIC procedure. To determine the probability of ketosis resolution, the logistic regression model
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included the farm, cow parity, enrollment week, treatment group (control, PG, and PG+CM), and
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interactions between these variables. To determine the probability of the occurrence of postpartum
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complications, the logistic regression model included the farm, calving season, cow parity, treatment
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group, and interactions between these variables. Backward stepwise regression was used in all models,
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and elimination was performed based on the Wald statistic criterion when P > 0.15. The odds ratio (OR)
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and 95% confidence interval (CI) were calculated. Data are presented as proportions and ORs with their
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respective 95% CIs.
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The blood BHBA, glucose and haptoglobin concentrations, RFS, and milk yield were analyzed using
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mixed models. The statistical models included group effects (control, PG, and PG+CM), cow parity
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(primiparous or multiparous), measuring time (Days 0, 3, 5, and 10), and two-way interactions between
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group, cow parity, and measuring time. Haptoglobin concentrations were not normally distributed;
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therefore, values were transformed to their natural logarithms for data analysis, although non-transformed
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data expressed as means and SEM are presented herein. For milk yield, the statistical models included
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group effects (control, PG, and PG+CM), measuring time (Days 30 and 60 postpartum), and two-way
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interactions between groups, cow parity, and measuring time. Cows were included in the model as a
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random effect. ANOVA followed by Duncan’s multiple range test was performed when either a group
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effect or an interaction between group and measuring time was observed. Loss of BCS from Day 0 to 10
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was analyzed by ANOVA followed by Duncan's multiple range test.
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Cox’s proportional hazard model with the PHREG procedure was used to analyze the chance of first
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insemination by 150 days and pregnancy by 210 days postpartum among the control, PG, and PG+CM
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groups. This estimated the chance of a cow having been inseminated or being pregnant at a given time.
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The time variables used in this model were the interval in days between calving and first insemination,
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and between calving and pregnancy. Cows that died, were sold, had not been inseminated by 150 days
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postpartum, or were not pregnant by 210 days postpartum were not included in the analysis. Cox models
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included farm, cow parity (primiparous or multiparous), enrollment week (1 or ≤2), and group (control,
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PG, and PG+CM). The proportional hazard rate was determined based on interactions between
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explanatory variables and time, and by evaluating Kaplan–Meier curves. The median and mean days to
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first insemination, or days to pregnancy, were determined by survival analysis using the Kaplan–Meier
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model and the LIFETEST procedure within SAS software. P < 0.05 was considered significant.
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3. Results
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The effect of the treatment protocols on the probability of resolution of ketosis on Days 3, 5, and 10
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was determined using the logistic regression model (Table 1). The probability of ketosis resolution was
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higher in the PG+CM group on Days 3 (OR: 6.3, P < 0.001), 5 (OR: 2.6, P < 0.05), and 10 (OR: 3.6, P <
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0.01), respectively, than in the control group, whereas the probability of resolution in the PG group was
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intermediate (P > 0.05). However, the logistic regression model revealed that the probability of resolution
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was not associated with farm, cow parity or enrollment week (P > 0.05).
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There was no effect of group on blood BHBA concentration (P > 0.05), but there were significant
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effects of measuring time (P < 0.0001) and an interaction between group and measuring time (P < 0.05)
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(Fig. 1). The BHBA concentration in the PG+CM group was lower than that of the control group at Days
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3 (P < 0.01) and 5 (P < 0.05), whereas the BHBA concentration in the PG group was intermediate (P >
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0.05). There was no parity effect on blood BHBA concentration. There was no effect of group on blood
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glucose (P > 0.05), but there were significant effects of measuring time (P < 0.01) and an interaction
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between group and measuring time (P = 0.05) (Fig. 2). The glucose concentrations in the PG and PG+CM
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group were higher than that of the control group at Day 3 (P < 0.01), while the glucose concentration in
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the PG+CM group was higher than that of the control group at Day 5 (P < 0.05). There was no parity
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effect on blood glucose concentration. There was a significant effect of measuring time on serum
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haptoglobin concentration (P < 0.0001), but no effect of group (ranges: 98.5 ± 32.3–120.0 ± 29.1 µg/mL)
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or any interaction between group and measuring time (P > 0.05) (data not shown). There was no effect of
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parity on serum haptoglobin concentration.
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There was a significant effect of group on RFS (P < 0.05), but there was no effect of measuring time
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or the interaction between group and measuring time (P > 0.05) (Fig. 3). The RFS in the PG group was
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higher than that of the control group on Day 5 (P < 0.05), whereas the RFSs in the PG and PG+CM
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groups were higher than that of the control group at Day 10 (P < 0.01). Multiparous cows had lower RFS
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than primiparous cows (P < 0.05). BCS loss from Day 0 to 10 in the control group (0.19 ± 0.02) was
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higher (P < 0.05) than those of the PG and PG+CM groups (each 0.12 ± 0.02).
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There were significant effects of group on milk yield (P < 0.05) and measuring time (P < 0.001), but
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no interaction between group and measuring time (P > 0.05) (Fig. 4). The average yield in the PG+CM
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group was greater than those of the control and PG groups on the 30th and 60th days postpartum (P <
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0.05). Primiparous cows showed lower milk yield than multiparous cows (P < 0.001). We determined the
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effect of treatment protocols for ketosis on the probability of occurrence of postpartum complications
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using the logistic regression model. Neither the treatment protocol (range: 48.0–54.0%), farm, calving
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season, and parity affected the incidence of postpartum complications (P > 0.05).
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The intervals between calving and first postpartum insemination or pregnancy did not differ among
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the groups (P > 0.05). The median and mean days to first postpartum insemination were 80 and 92.6 ± 4.7
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in the control group, 84 and 99.1 ± 5.5 in the PG group, and 97 and 103.1 ± 4.7 in the PG+CM group,
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respectively. The median and mean days to pregnancy were 154 and 152.4 ± 7.3 in the control group, 160
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and 154.7 ± 8.7 in the PG group, and 169 and 156.3 ± 8.1 in the PG+CM group, respectively.
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4. Discussion
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We have evaluated the effect of treatment with PG or PG plus an L-carnitine and methionine-
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containing product (Metabolase®, Fatro, Bologna, Italy) on the resolution of ketosis, blood BHBA,
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glucose, and haptoglobin concentrations, postpartum health (RFS, BCS loss, and complications), milk
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yield, and reproductive performance in dairy cows. Treatment with PG plus L-carnitine and methionine
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resulted in faster resolution of ketosis, a lower BHBA and higher glucose concentrations, a higher RFS,
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and lower BCS loss, resulting in higher milk yield, while it did not affect serum haptoglobin
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concentration, incidence of postpartum complications, or reproductive performance.
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Cows treated with PG plus L-carnitine and methionine had a higher probability of ketosis resolution
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on Days 3, 5, and 10, whereas those that received only PG were not more likely to recover from ketosis
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than controls on this timescale. The higher probability of ketosis resolution in PG+CM cows was
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associated with improvements in other indicators of health, including lower blood BHBA and higher
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glucose concentrations, higher RFS, and less BCS loss during the study period. It is likely that the
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supplementation of L-carnitine and methionine, in addition to PG, not only increased hepatic glucose
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production by stimulating metabolite flux through pyruvate carboxylase, but also enhanced the capacity
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of the liver to export triacylglycerol in the form of VLDL [31,33,35].
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Fatty infiltration represents a harmful stimulus to liver parenchymal cells, and consequently
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production of haptoglobin, acute phase protein, may be higher in affected hepatocytes [30]. Consistent
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with this, a previous study showed that haptoglobin increased in cows with ketosis (by 4–6 fold)
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compared with their healthy counterparts during the transition period [44]. However, we found no effect
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of either PG or PG plus L-carnitine and methionine on serum haptoglobin concentration. This may be
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because postpartum disorders are often associated with acute inflammatory processes, including dystocia,
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retained placenta, or metritis, which would confound the interpretation of haptoglobin concentration
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among the groups in the present study. Indeed, several previous studies have demonstrated that such
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postpartum disorders were associated with elevated haptoglobin concentrations [45–47]. However, other
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studies have shown that oral PG increases blood glucose and insulin, but decreases BHBA and NEFA
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concentrations [24,48]. Moreover, improved resolution of ketosis and lower incidence of postpartum
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disease (abomasal displacement) and culling [9,11] have also been reported, which are not consistent with
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our data. The reason for the discrepancies among these studies is not clear; therefore, further work is
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required to resolve these. Monitoring of the changes in feed intake during the periparturient period is very important in dairy
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cows because suboptimal DMI is associated with several postpartum disorders, including ketosis, metritis,
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and mastitis [49–51]. However, the monitoring of DMI for individual cows requires enormous labor and
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is impractical for commercial dairy farms. Thus, the measurement of rumen fill is used as an indirect tool
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for the evaluation of feed intake status. This is a relatively simple method that is feasible for commercial
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dairy farms to undertake [37,52]. In addition, BCS is a simple, indirect assessment tool for the monitoring
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of energy balance. BCS at calving and the change in BCS during the dry period were associated with the
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incidence of postpartum diseases and subsequent reproductive performance in dairy cows [53–55].
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Therefore, we used RFS and the loss of BCS as indicators for feed intake status and energy balance in the
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present study.
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Higher RFS in the PG and PG+CM groups than in the control group at Day 10 imply that cows that
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received two treatments had eaten more than the non-treated control cows. In addition, lower loss of BCS
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during the experimental period (Days 0–10) in the two treatment groups suggests that the extent of their
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NEB had been alleviated by treatment with PG or PG plus L-carnitine and methionine. Taken together,
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our data indicate that a higher RFS and lower BCS loss, as well as decreased blood BHBA and increased
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glucose concentrations, which may imply higher feed intake and lower NEB, might contribute to a higher
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incidence of ketosis resolution in cows that receive PG plus L-carnitine and methionine.
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Previous publications have shown decreases in milk production in cows with ketosis [8,9,56]. Thus,
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early and effective treatment for ketosis may prevent a subsequent decrease in milk production. Cows
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treated with PG plus L-carnitine and methionine had higher milk yields at Days 30 and 60 postpartum
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than those treated with only PG, or controls, in this study. The positive effect on milk yield in the PG+CM
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group might be a consequence of the earlier resolution of ketosis and better health status compared to the
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other two groups, reflected in higher RFS and lower loss of BCS. Moreover, our results are consistent
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with a previous study in which milk yield was similar between cows that received PG orally and untreated
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control cows [22]. However, another previous study reported increased milk yield in cows treated with
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300 mL PG orally in the first month of lactation [11], which is inconsistent with our data. We found no effects of PG or PG plus L-carnitine and methionine on the incidence of postpartum
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complications and reproductive outcomes (intervals from calving to first postpartum insemination and
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pregnancy). Our results are broadly similar to those of a previous study, in which oral PG administration
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did not affect the incidence of clinical ketosis compared to untreated control cows [22]. Inconsistent with
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our data, oral PG administration decreased the risk of developing an abomasal displacement in a previous
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study [9]. However, this study found no difference in the calving to conception interval between cows that
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received PG and untreated cows [9], which is similar to our results.
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Taken together, our data show that cows treated with PG plus L-carnitine and methionine for ketosis
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resulted in earlier resolution and higher milk yield compared to untreated controls and cows treated with
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PG only. This could be attributed to the provision of a better glucose substrate and the reduction of
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ketogenesis in the liver, as suggested in the Introduction. Moreover, the beneficial outcomes described in
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the present study might also be due to the increase in postpartum health resulting from increased feed
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intake and lower NEB, as demonstrated not only by the higher RFS and lower loss of BCS, but also by
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the decreased blood BHBA and increased glucose concentrations. However, we found no effects of
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treatment with PG plus L-carnitine and methionine on the incidence of postpartum complications and
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reproductive outcomes. The results showing earlier resolution from ketosis and higher milk yield in cows
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treated with PG plus L-carnitine and methionine might be helpful in guiding treatments for ketotic dairy
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cows, which in turn could potentially mitigate the economic loss associated with ketosis.
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Acknowledgments
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This work was carried out with the support of the “Cooperative Research Program for Agriculture
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Science & Technology Development (Project No. PJ01081802)” Rural Development Administration,
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Republic of Korea.
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Conflicts of interest
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None.
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Figure captions
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Figure 1. Mean (± SEM) blood β-hydroxybutyrate (BHBA) concentrations in the control, PG (propylene
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glycol), and PG+CM (L-carnitine and methionine) group cows (n = 50 per group) at Days 0, 3, 5, and 10
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after treatment. G: group effect, Day: measuring time effect. G*Day: group-by-measuring time effect.
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a,b
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superscripts differ with P < 0.01 among groups.
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c,d
Means with different
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Means with different superscripts differ with P < 0.05 among groups.
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Figure 2. Mean (± SEM) blood glucose concentrations in the control, PG, and PG+CM group cows (n =
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50 per group) at Days 0, 3, 5, and 10 after treatment. G: group effect, Day: measuring time effect. G*Day:
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group-by-measuring time effect.
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c,d
a,b
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Means with different superscripts differ with P < 0.05 among groups.
Means with different superscripts differ with P < 0.01 among groups.
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Figure 3. Mean (± SEM) rumen fill scores in the control, PG, and PG+CM group cows (n = 50 per group)
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at Days 0, 3, 5, and 10 after treatment. G: group effect, Day: measuring time effect. G*Day: group-by-
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measuring time effect.
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with different superscripts differ with P < 0.01 among groups.
c,d
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a,b
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Figure 4. Mean (± SEM) milk yield in the control (n = 49), PG (n = 48), and PG+CM (n = 48) groups at
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the 30th and 60th days postpartum. G: group effect, Day: measuring time effect. G*Day: group-by-
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measuring time effect. a,bMeans with different superscripts differ with P < 0.05 among groups.
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Table 1. The effect of treatment protocols for ketosis on the probability of resolution of ketosis on Days 3,
527
5, and 10 determined using a logistic regression model. Variable
Resolution a
evaluation
percentage (n )
b
Control
12.0% (6/50)
Reference
PG
22.0% (11/50)
2.1
PG+CM
46.0% (23/50)
6.3
Farm d
Cow parity
e
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Enrollment week Group
531 532
> 0.05
0.509–2.925
> 0.05
PG+CM
48.0% (24/50)
2.6
1.133–6.090
< 0.05
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Group
> 0.05 > 0.05 > 0.05
Reference
44.0% (22/50)
2.2
0.920–5.148
> 0.05
56.0% (28/50)
3.6
1.496–8.464
< 0.01
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30.0% (15/50)
> 0.05
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Farm
c
> 0.05
1.2
PG+CM
530
> 0.05
30.0% (15/50)
PG
Cow parity
> 0.05
Enrollment week
> 0.05
, cases/group total.
b
< 0.001
PG
Control
529
2.256–17.291
Reference
Enrollment week
a
> 0.05
26.0% (13/50)
Cow parity
528
0.699–6.115
Control
Farm
Day 10
P-value
OR
Group
Day 5
95% CI
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Day 3
c
Adjusted
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Days of
, odds ratio.
, confidence interval.
d
, categorized as either primiparous or multiparous.
e
, divided into 1 or ≤2.
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Highlights
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- We determined the effect of ketosis treatment with propylene glycol (PG) or PG plus L-carnitine and
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methionine.
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- PG plus L-carnitine and methionine improved the chances of resolution of ketosis and increased milk
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yield.
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- Outcomes might be associated with better postpartum health due to increased feed intake and lower
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NEB.
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